Page 1 of 69 

SECOND AMENDED FINAL REPORT

Study Title

Bacterial Reverse Mutation Assay Using Gas-Phase Exposure 

Test Article

Authors

Valentine O. Wagner, III, M.S. 

Renee M. Hines, B.S. 

Meena Jois, B.S. 

Study Completion Date

Original Date: 04 September 2008 

Amended Final Report 1 Date: 11 January 2011 

Amended Final Report 2 Date: 27 April 2011 

Testing Facility

BioReliance 

9630 Medical Center Drive 

Rockville, MD 20850 

BioReliance Study Number

AC12HE.502700.BTL 

Sponsor




BioReliance 

Study No. AC12HE.502700.BTL 6 

Bacterial Reverse Mutation Assay Using Gas-Phase Exposure 

STUDY INFORMATION 

Sponsor: 

Study Representative/Study Monitor: 

Testing Facility: BioReliance 

9630 Medical Center Drive 

Rockville, Maryland 20850 

Test Article I.D.: 

Synonyms: 

Test Article CAS No.: 

BioReliance Study No.: AC12HE.502700.BTL 

Test Article Purity: 99.978 area% 

Test Article Lot No.: 40883-50 (Sample 0001) 

Batch#1-Cyl#1 (Sample 0002) 

Test Article CAS No.: 

Test Article Description: Clear, liquefied gas 

Storage Conditions: Room temperature, upright in the dark without 

desiccant 

Test Article Receipt and Login: 22 February 2008 (Sample 0001) 

15 July 2010 (Sample 0002) 

Study Initiation: 04 April 2008 

Experimental Start Date: 09 April 2008 

Experimental Completion Date: 06 October 2010 

Laboratory Managers: Melissa R. VanDyke, B.S. (prior to January 2009)

Emily W. Dakoulas, B.S. (effective January 2009) 
BioReliance 

Study No. AC12HE.502700.BTL 7 

TABLE OF CONTENTS 

Page 

SUMMARY.............................................................................................................................. 8

PURPOSE............................................................................................................................... 10

CHARACTERIZATION OF TEST AND CONTROL ARTICLES...................................... 10

MATERIALS AND METHODS............................................................................................ 12

RESULTS AND DISCUSSION............................................................................................. 18

CONCLUSION....................................................................................................................... 19

REFERENCES ....................................................................................................................... 19 

DATA TABLES ..................................................................................................................... 21 

APPENDIX I: Historical Control Data................................................................................... 42

APPENDIX II: Study Protocol ............................................................................................... 45

APPENDIX III: Information for Japanese Regulatory Agencies........................................... 61

APPENDIX IV: Test Article Characterization....................................................................... 67
BioReliance 

Study No. AC12HE.502700.BTL 8 

SUMMARY 

The test article, was tested in the Bacterial Reverse Mutation Assay Using 

Gas-Phase Exposure using Salmonella typhimurium tester strains tester strains TA98, TA100, 

TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence 

of Aroclor-induced rat liver S9. The assay was performed in three phases, using the desiccator 

method, a modification of the plate incorporation method. The first phase, the preliminary 

toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second 

phase, the mutagenicity assay was used to evaluate the mutagenic potential of the test article. 

The third phase, the confirmatory mutagenicity assay was used to evaluate and confirm the 

mutagenic potential of the test article. 

Ambient air was used as the negative control. 

In the preliminary toxicity assay, the maximum dose tested was 37 mmoles/L. The dose levels 

tested were 1, 3, 6, 8, 11, 21 and 37 mmoles/L (24,500; 73,400; 147,000; 196,000, 269,000; 

513,000 and 905,000 ppm at 25°C, respectively). The test system was exposed to the test article 

in the desiccators for 24 hours. No precipitate was observed. No background lawn toxicity was 

observed however a reduction in revertant counts was observed beginning at 11, 21 or at 

37 mmoles/L with some test conditions. Based on the findings of the preliminary toxicity assay, 

the maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L. 

In the mutagenicity assay, no positive mutagenic response was observed. The dose levels tested 

were 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,000; 196,000, 269,000; 513,000 and 

905,000 ppm at 25°C, respectively). The test system was exposed to the test article in the 

desiccators for 48 hours. No precipitate was observed. Toxicity was observed beginning at 11, 

21 or at 37 mmoles/L. A confirmatory mutagenicity assay was conducted at the Sponsor’s 

request to meet the requirements for Japanese regulatory submission. 

The confirmatory mutagenicity assay was conducted at dose levels of 3, 6, 8, 11, 21 and 

37 mmoles/L (73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm at 25°C, 

respectively). No positive mutagenic response was observed with tester strains TA100, TA1535 

and WP2 uvrA in the absence of S9 activation and with tester strains TA100, TA1535 and 

TA1537 in the presence of S9 activation. No precipitate was observed. Toxicity was observed 

beginning at 21 or 37 mmoles/L. Tester strain TA98 in the presence and absence of S9 was not 

evaluated due to confluent growth but was retested. Due to excessive toxicity, as a result of a 

drop in the revertant counts beginning at 8 mmoles/L, tester strain TA 1537 in the absence of S9 

was retested. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the 

absence of S9 activation was not evaluated but was retested. 

In the retest of the confirmatory mutagenicity assay, the dose levels tested were 0.7, 1, 3, 6, 8, 

11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 

905,000 ppm at 25°C, respectively). No positive responses were observed with tester strains 

TA98 and TA1537 in the absence of S9 activation and with tester strains TA98 and WP2 uvrA 
BioReliance 

Study No. AC12HE.502700.BTL 9 

in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning 

at 21 or 37 mmoles/L. 

The results of the Bacterial Reverse Mutation Test Using Gas-Phase Exposure indicate that, 

under the conditions of this study, test article did not cause a positive mutagenic 

response in either the presence or absence of Aroclor-induced rat liver S9. 
BioReliance 

Study No. AC12HE.502700.BTL 10 

PURPOSE 

The purpose of this study was to evaluate the mutagenic potential of the test article gas by 

measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella

typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence 

and absence of Aroclor-induced rat liver S9. A copy of the Historical Negative and Positive 

Control Values is included in Appendix I. Copies of the study protocol and amendments are 

included in Appendix II. 

This study was conducted in compliance with the testing guideline of the OECD (1998) and 

Japanese guidelines Official notice of J MHLW, METI and ME (21 November 2003): 

YAKUSHOKUHATSU No.1121002 SEIKYOKU No.2 KANPOKIHATSU No. 031121002. 

CHARACTERIZATION OF TEST AND CONTROL ARTICLES 

The test article, was received by BioReliance on 22 February 2008 and was 

assigned the code number AC12HE. Sample 0002 of the test article was received on 15 July 

2010. The test article was characterized on the Material Safety Data Sheet as a clear volatile 

liquid. Per the protocol, the test article should be stored at ambient conditions in its shipping 

cylinder. Upon arrival at BioReliance, the test article was stored upright at room temperature in 

the dark without desiccant. 

The Sponsor has determined the identity, strength, purity, composition or other characteristics to 

define lot 40883-50 and batch#1 cylinder#1 of the test article. Copies of the Test Article 

Characterization for each lot/batch are included in Appendix IV. 

The test article was supplied in a gas cylinder. All dosing was at room temperature under 

yellow light. 

The positive control articles have been characterized as per the Certificates of Analysis on file 

with the testing facility. The stability of the negative and positive control articles and their 

mixtures was demonstrated by acceptable results that met the criteria for a valid test. 

Positive controls plated concurrently with the mutagenicity assay are listed in the following 

table. All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, 

which was diluted in water. All subdivided solutions of positive control were stored at -5 to 

-30°C. 
BioReliance 

Study No. AC12HE.502700.BTL 11 

Strain S9 

Activation Positive Control Concentration (μg/plate) 

All Salmonella Strains 1.0 (B1)

WP2 uvrA 

2-aminoanthracene 

CAS No. 613-13-8 

(Aldrich Chemical Co., Inc.) 

Lot No. 12317CE 

Exp. Date 01-Feb-2009 

Purity 99.9% 

(Sigma Aldrich Chemical Co.) 

Lot No. 03403ED 

Exp. Date 22-Jan-2012 

Purity 99.8% 

10 

TA100 

Rat 

(Sigma Aldrich Chemical Co.) 

Lot No. 03403ED 

Exp. Date 22-Jan-2012 

Purity 99.8% 

2.0 (B2)

TA98 

2-nitrofluorene 

CAS No. 607-57-8 

(Aldrich Chemical Co., Inc.) 

Lot No. 03926DC 

Exp. Date 18-Aug-2010 

Purity 98.1% 

(Sigma Aldrich Chemical Co.) 

Lot No. 03319JD 

Exp. Date 28-Feb-2011 

Purity 98.1% 

1.0 

TA100, TA1535 

None 

Sodium azide 

CAS No. 26628-22-8 

(Sigma Chemical Co.) 

Lot No. 073K0119 

Exp. Date 31-Jul-2008 

Purity 99.9% 

(Sigma Aldrich Chemical Co.) 

Lot No.71980 

Exp. Date 28-Dec-2010 

Purity 99.8% 

1.0 
BioReliance 

Study No. AC12HE.502700.BTL 12 

Strain S9 

Activation Positive Control Concentration (μg/plate) 

TA1537 

9-aminoacridine 

(Sigma Chemical Co.) 

Lot No. 106F06682 

Exp. Date 08-Nov-2009 or 

30-Oct-2010 

CAS No. 90-45-9 

Purity >97% 

75 

WP2 uvrA 

Methyl methanesulfonate 

CAS No. 66-27-3 

(Aldrich Chemical Co., Inc.) 

Lot No. 126K3721 

Exp. Date 07-Jan-2011 

Purity >99.9% 

(Sigma Aldrich Chemical Co.) 

Lot No.76296KJ 

Exp. Date 02-Jun-2012 

 Purity 99.8% 

1,000 

MATERIALS AND METHODS 

For submission to Japanese regulatory agencies, additional information is included in 

Appendix III. 

Test System 

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, 

TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as 

described by Green and Muriel (1976). Salmonella tester strains were received from Dr. Bruce 

Ames’ designated distributor, Discovery Partners International, San Diego, California. E. coli

tester strains were received from the National Collection of Industrial and Marine Bacteria, 

Aberdeen, Scotland. The tester strains were also obtained from Molecular Toxicology Inc. 

(MolTox) using cultures derived from the above sources. 

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to 

histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted 

by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that 

cause both frameshift and basepair substitution mutations. Specificity of the reversion 

mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift 

mutations (Green and Muriel, 1976). 
BioReliance 

Study No. AC12HE.502700.BTL 13 

Each tester strain culture was inoculated from the appropriate master plate, from the appropriate 

frozen stock or with the appropriate lyophilized pellet(s). To ensure that cultures were harvested 

in late log phase, the length of incubation was controlled and monitored. At the end of the 

working day, each inoculated flask was placed in a shaker/incubator programmed to begin 

shaking at approximately 125 to 175 rpm and incubating at 37±2°C for approximately 12 to 

14 hours before the anticipated time of harvest. Each culture was monitored 

spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer 

of approximately 109

 cells per milliliter. The actual titers were determined by viable count 

assays on nutrient agar plates. 

Metabolic Activation System 

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was 

prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of 

Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The lots of S9 were prepared by and 

purchased from MolTox (Boone, NC) and stored at -60°C or colder until used. Each bulk 

preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms 

mutagenic to Salmonella typhimurium TA100. The S9 homogenate was used within 6 months 

after preparation. 

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM 

glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 

33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), 

containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To 

confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective 

agar. 

Preliminary Toxicity Assay 

The preliminary toxicity assay was used to establish the dose-range over which the test article 

would be assayed. Negative (air) control and seven dose levels of the test article were plated, 

one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA 

on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. 

Mutagenicity Assay 

The mutagenicity assay was used to evaluate the mutagenic potential of the test article. Six dose 

levels of test article along with appropriate negative (air) control and positive controls were 

plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective 

minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test 

article, vehicle control and positive controls were plated in triplicate. 
BioReliance 

Study No. AC12HE.502700.BTL 14 

Confirmatory Mutagenicity Assay 

The mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test 

article. Six or eight dose levels of test article along with appropriate negative (air) control and 

positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and 

WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver 

S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate. 

Plating and Scoring Procedures 

The test system was exposed to the test article via the desiccator methodology, a modification of 

the plate incorporation methodology originally described by Ames et al. (1975) and updated by 

Maron and Ames (1983). This test system has been shown to detect a wide range of classes of 

chemical mutagens (McCann et al., 1975; McCann and Ames, 1976). The desiccator 

methodology has been shown to be an effective method for detecting the genotoxic activity of 

volatile and gaseous test articles (Wagner et al., 1992). 

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), 

was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final 

concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 

25 mL of water for each 100 mL of minimal top agar. For the preparation of media and 

reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner 

minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom 

agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented 

with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner 

salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). 

Each plate was labeled with a code system that identified the test article, test phase, dose level, 

tester strain and activation, as described in detail in BioReliance's Standard Operating 

Procedures. 

For the positive controls, one-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain 

(cells seeded) and 50 μL aliquot of appropriate positive control were added to 2.0 mL of molten 

selective top agar at 45±2°C. When plating negative (air) control, the addition of positive 

control was omitted. After vortexing, the mixture was overlaid onto the surface of 25 mL of 

minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated 

for approximately 48 to 72 hours at 37±2°C. The negative control plates were placed inside of a 

desiccator for the duration of the incubation period. Plates that were not counted immediately 

following the incubation period were stored at 2-8°C until colony counting could be conducted. 

For test article, one-half (0.5) milliliter of S9 or Sham mix and 100 μL of tester strain (cells 

seeded) were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the 

mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had 

solidified, the plates were inverted and placed uncovered in the appropriate number of 9-liter 

desiccators. An appropriate quantity of the test article was introduced into each desiccator by 
BioReliance 

Study No. AC12HE.502700.BTL 15 

withdrawing an appropriate amount of air and replacing it with the test article. The desiccators 

were incubated, with stirring, for approximately 24 hours (Experiment A1) or 48 hours 

(Experiments B1, B2 and B3) at 37±2°C. Following the 24-hour incubation in Experiment A1, 

the plates were removed from the desiccators and incubated with the lids replaced at 37±2°C for 

an additional 24 hours. When plating the untreated controls, one-half (0.5) milliliter of S9 or 

Sham mix and 100 μL of tester strain were added to 2.0 mL of molten selective top agar at 

45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal 

bottom agar. After the overlay had solidified, the plates were inverted and incubated under the 

same conditions as the assay plates. When plating the positive controls, one-half (0.5) milliliter 

of S9 or Sham mix, 100 μL of tester strain and a 50 μL aliquot of appropriate positive control 

were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was 

overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the 

plates were inverted and incubated under the same conditions as the assay plates. Plates that 

were not counted immediately following the incubation period were stored at 2-8°C until colony 

counting could be conducted. 

The condition of the bacterial background lawn was evaluated for evidence of test article 

toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination 

without magnification. Toxicity and degree of precipitation were scored relative to the vehicle 

control plate using the codes shown in the following table. 

Code Description Characteristics 

1 or no 

code Normal Distinguished by a healthy microcolony lawn. 

2 Slightly 

Reduced 

Distinguished by a noticeable thinning of the microcolony lawn 

and possibly a slight increase in the size of the microcolonies 

compared to the vehicle control plate. 

3 Moderately 

Reduced 

Distinguished by a marked thinning of the microcolony lawn 

resulting in a pronounced increase in the size of the microcolonies 

compared to the vehicle control plate. 

4 Extremely 

Reduced 

Distinguished by an extreme thinning of the microcolony lawn 

resulting in an increase in the size of the microcolonies compared 

to the vehicle control plate such that the microcolony lawn is 

visible to the unaided eye as isolated colonies. 

5 Absent Distinguished by a complete lack of any microcolony lawn over 

greater than or equal to 90% of the plate. 

6 Obscured by 

Particulate 

The background bacterial lawn cannot be accurately evaluated due 

to microscopic test article particulate. 

NP Non-Interfering 

Precipitate 

Distinguished by precipitate on the plate that is visible to the 

naked eye but any precipitate particles detected by the automated 

colony counter total less than or equal to 10% of the revertant 

colony count (e.g., less than or equal to 3 particles on a plate with 

30 revertants). 
BioReliance 

Study No. AC12HE.502700.BTL 16 

Code Description Characteristics 

IP Interfering 

Precipitate 

Distinguished by precipitate on the plate that is visible to the 

naked eye and any precipitate particles detected by the automated 

colony counter exceed 10% of the revertant colony count (e.g., 

greater than 3 particles on a plate with 30 revertants). These plates 

are counted manually. 

Revertant colonies for a given tester strain and activation condition, except for positive controls, 

were counted either entirely by automated colony counter or entirely by hand unless the plate 

exhibited toxicity. 

Evaluation of Results 

For each replicate plating, the mean and standard deviation of the number of revertants per plate 

were calculated and are reported. 

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean 

revertants at the peak of the dose response was equal to or greater than 3.0-times the mean 

vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged 

positive if the increase in mean revertants at the peak of the dose response was equal to or 

greater than 2.0-times the mean vehicle control value. 

An equivocal response is a biologically relevant increase in a revertant count that partially meets 

the criteria for evaluation as positive. This could be a dose-responsive increase that does not 

achieve the respective threshold cited above or a non-dose responsive increase that is equal to or 

greater than the respective threshold cited. A response will be evaluated as negative, if it is 

neither positive nor equivocal. 

Criteria for a Valid Test 

The following criteria must be met for the initial toxicity-mutation and the confirmatory 

mutagenicity assays to be considered valid. All Salmonella tester strain cultures must 

demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. 

Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 

plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All 

cultures must demonstrate the characteristic mean number of spontaneous revertants in the 

vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; 

TA1537, 3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, 

tester strain culture titers must be greater than or equal to 0.3x109

 cells/mL. The mean of each 

positive control must exhibit at least a 3.0-fold increase in the number of revertants over the 

mean value of the respective vehicle control. A minimum of three non-toxic dose levels is 

required to evaluate assay data. A dose level is considered toxic if one or both of the following 

criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared 

to the mean vehicle control value. This reduction must be accompanied by an abrupt 
BioReliance 

Study No. AC12HE.502700.BTL 17 

dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background 

lawn (background code 3, 4 or 5). 

Automated Data Collection Systems 

The primary computer applications used for the collection of data include: 

Minicount Colony Counter (Imaging Products International), Sorcerer Colony Counter and 

Ames Study Manager (Perceptive Instruments), LIMS System (BioReliance), Excel 2003 

(Microsoft Corporation) and Kaye Lab Watch Monitoring System (Kaye GE). 

Archives 

All raw data, the protocol and all reports for procedures performed at BioReliance will be 

maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA 

unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Per this SOP, 

paper records will be retained for at least three years after which time the Sponsor will be 

contacted for a decision as to the final disposition of the materials. All study materials returned 

to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReliance 

archives for a minimum of 10 years. Raw data, the protocol and reports generated at facilities 

other than BioReliance will be archived per the contractual arrangements between the facility 

and the Sponsor. 

Deviations 

The following deviation occurred during the conduct of this study: 

In deviation from the study protocol, the assay plates for Experiment B1 were incubated in the 

desiccators for approximately 48 hours as opposed to 24 hours as stated in the protocol and as 

requested by the Sponsor (Event No. 4732). The Sponsor requested the 24 hour exposure to be 

consistent with work on fluorocarbons conducted over the past 20 years. Since the longer 

exposure time demonstrated definitive toxicity, this demonstrates exposure of the test system to 

the test article and provides greater confidence of the negative result. Therefore, the Study 

Director has concluded that this deviation did not adversely impact the integrity of the data or 

the validity of the study conclusion. 
BioReliance 

Study No. AC12HE.502700.BTL 18 

RESULTS AND DISCUSSION 

Sterility Results 

No contaminant colonies were observed on the sterility plates for the S9 and Sham mixes.

Preliminary Toxicity Assay 

The results of the preliminary toxicity assay are presented in Table 1 through Table 5. In the 

preliminary toxicity assay, the maximum dose tested was 37 mmoles/L. The dose levels tested 

were 1, 3, 6, 8, 11, 21 and 37 mmoles/L (24,500; 73,400; 147,000; 196,000, 269,000; 513,000 

and 905,000 ppm at 25°C, respectively). The test system was exposed to the test article in the 

desiccators for 24 hours. No background lawn toxicity was observed however a reduction in 

revertant counts was observed beginning at 11, 21 or at 37 mmoles/L with some test conditions. 

Based on the findings of the preliminary toxicity assay, the maximum dose plated in the 

confirmatory mutagenicity assay was 37 mmoles/L. 

Tester Strain Titer Results 

Tester Strain 

Experiment TA98 TA100 TA1535 TA1537 WP2 uvrA 

Titer Value (x 109

 cells per mL) 

B1 0.8 0.7 1.0 0.7 1.7 

B2 -- 0.3 0.7 1.0 5.6 

B3 1.1 -- -- 0.9 3.0 

Mutagenicity Assay 

The results of the mutagenicity assay are presented in Table 6 through Table 15 and 

summarized in Table 16. These data were generated in Experiment B1. 

The dose levels tested were 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,000; 196,000, 

269,000; 513,000 and 905,000 ppm at 25°C, respectively). The test system was exposed to the 

test article in the desiccators for 48 hours. No positive mutagenic responses were observed with 

any of the tester strains in the presence or absence of S9 activation. No precipitate was 

observed. Toxicity was observed beginning at 11, 21 or at 37 mmoles/L. 

Confirmatory Mutagenicity Assay 

The results of the confirmatory mutagenicity assay are presented in Table 17 through Table 20. 

These data were generated in Experiments B2 and B3. 
BioReliance 

Study No. AC12HE.502700.BTL 19 

The dose levels tested were 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,000; 196,000, 

269,000; 513,000 and 905,000 ppm at 25°C, respectively). No positive mutagenic response was 

observed with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation and 

with tester strains TA100, TA1535 and TA1537 in the presence of S9 activation. No precipitate 

was observed. Toxicity was observed beginning at 21 or 37 mmoles/L. Tester strain TA98 in 

the presence and absence of S9 was not evaluated due to confluent growth but was retested. 

Due to excessive toxicity, as a result of a drop in the revertant counts beginning at 8 mmoles/L, 

tester strain TA 1537 in the absence of S9 was retested. Due to an unacceptable vehicle control 

value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated but was retested. 

In the retest of the confirmatory mutagenicity assay, the dose levels tested were 0.7, 1, 3, 6, 8, 

11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 

905,000 ppm at 25°C, respectively). No positive responses were observed with tester strains 

TA98 and TA1537 in the absence of S9 activation and with tester strains TA98 and WP2 uvrA 

in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning 

at 21 or 37 mmoles/L. 

CONCLUSION 

All criteria for a valid study were met as described in the protocol. The results of the Bacterial 

Reverse Mutation Assay Using Gas-Phase Exposure indicate that, under the conditions of this 

study, did not cause a positive mutagenic response in either the presence or absence 

of Aroclor-induced rat liver S9. 

Stability data were not provided for either lot/batch of the test article. In the absence of stability 

data, the Study Director has concluded that the test article was negative with the caveat that the 

maximum required concentration may not have been achieved due to instability or impurities. 

At the request of the Sponsor, dosing formulation analyses for concentration and stability were 

not conducted. Due to the lack of dose formulation analysis, the interpretation of the study data 

were based on the nominal dose levels as documented in the study records and not on the actual 

formulated test article concentrations as confirmed by analytical analysis. Nevertheless, toxicity 

in the assay demonstrated that the test system was dosed up to the regulatory required level. 

REFERENCES 

Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and 

Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test, Mutation Research, 

31:347-364. 

Green, M.H.L. and W.J. Muriel (1976) Mutagen testing using trp+ reversion in Escherichia coli, 

Mutation Research 38:3-32. 
BioReliance 

Study No. AC12HE.502700.BTL 20 

Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, 

Mutation Research, 113:173-215. 

McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the 

Salmonella/microsome test: assay of 300 chemicals; discussion. Proc. Natl. Acad. Sci. USA 

73:950-954. 

McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as 

mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 

72:5135-5139. 

OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum 

to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 

1998. 

Wagner, III, V.O., San, R.H.C. and Zeiger, E. (1992) Desiccator Methodology for Salmonella

Mutagenicity Assay of Vapor-Phase and Gas-Phase Test Materials. Environmental and 

Molecular Mutagenesis 19:68. 

Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G. (1990). Comparison of Salmonella

typhimurium (TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. 

Vogel, H.J. and D.M. Bonner (1956) Acetylornithinase of E. coli: Partial Purification and Some 

Properties, J. Biol. Chem., 218:97-106. 
BioReliance 

Study No. AC12HE.502700.BTL 21 

Bacterial Mutation Assay 

Table 1 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA98 Experiment No : A1 

Vehicle : Air Control Date Plated : 9-Apr-08 

Concentration 

mmoles/L Without S9 Revertants per plate 

Background 

Code 

With S9 Revertants 

per plate Background Code 

Vehicle 10 1 10 1 

1 10 1 11 1 

3 9 1 12 1 

6 10 1 11 1 

8 11 1 17 1 

11 11 1 13 1 

21 11 1 12 1 

37 5 2 3 2 

 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 22 

Bacterial Mutation Assay 

Table 2 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA100 Experiment No : A1 

Vehicle : Air Control Date Plated : 9-Apr-08 

Concentration 

mmoles/L Without S9 Revertants per plate 

Background 

Code 

With S9 Revertants 

per plate Background Code 

Vehicle 202 1 219 1 

1 206 1 207 1 

3 238 1 197 1 

6 241 1 180 1 

8 244 1 197 1 

11 265 1 229 1 

21 133 1 198 1 

37 263 1 228 2 

 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 23 

Bacterial Mutation Assay 

Table 3 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1535 Experiment No : A1 

Vehicle : Air Control Date Plated : 9-Apr-08 

Concentration 

mmoles/L Without S9 Revertants per plate 

Background 

Code 

With S9 Revertants 

per plate Background Code 

Vehicle 7 1 13 1 

1 12 1 8 1 

3 13 1 12 1 

6 11 1 11 1 

8 11 1 9 1 

11 13 1 12 1 

21 13 1 6 1 

37 8 1 5 1 

 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 24 

Bacterial Mutation Assay 

Table 4 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1537 Experiment No : A1 

Vehicle : Air Control Date Plated : 9-Apr-08 

Concentration 

mmoles/L Without S9 Revertants per plate 

Background 

Code 

With S9 Revertants 

per plate Background Code 

Vehicle 8 1 11 1 

1 7 1 8 1 

3 8 1 10 1 

6 9 1 5 1 

8 6 1 8 1 

11 7 1 3 1 

21 7 1 4 1 

37 6 2 4 2 

 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 25 

Bacterial Mutation Assay 

Table 5 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : WP2 uvrA Experiment No : A1 

Vehicle : Air Control Date Plated : 9-Apr-08 

Concentration 

mmoles/L Without S9 Revertants per plate 

Background 

Code 

With S9 Revertants 

per plate Background Code 

Vehicle 17 1 18 1 

1 19 1 16 1 

3 17 1 17 1 

6 C NA 19 1 

8 17 1 14 1 

11 15 1 15 1 

21 12 1 16 1 

37 3 1 12 1 

 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 

C=Contaminated 

NA= Not Applicable 
BioReliance 

Study No. AC12HE.502700.BTL 26 

Bacterial Mutation Assay 

Table 6 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA98 Experiment No : B1 

Activation Condition : None Cells Seeded : 0.8 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 26 1 

2 45 1 

3 35 1 35 10 

3 1 20 1 

2 25 1 

3 35 1 27 8 

6 1 35 1 

2 20 1 

3 30 1 28 8 

8 1 25 1 

2 25 1 

3 15 1 22 6 

11 1 22 1 

2 20 1 

3 16 1 19 3 

21 1 4 1 

2 4 1 

3 3 1 4 1 

37 1 0 4 

2 0 4 

3 0 4 0 0 

Positive Control 2-nitrofluorene 1.0 μg per plate 

1 162 1 

2 158 1 

 3 67 1 129 54 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 27 

Bacterial Mutation Assay 

Table 7 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA98 Experiment No : B1 

Activation Condition : Rat Liver S9 Cells Seeded : 0.8 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 32 1 

2 21 1 

3 20 1 24 7 

3 1 17 1 

2 14 1 

3 14 1 15 2 

6 1 25 1 

2 13 1 

3 38 1 25 13 

8 1 17 1 

2 15 1 

3 21 1 18 3 

11 1 14 1 

2 9 1 

3 15 1 13 3 

21 1 2 1 

2 6 1 

3 2 1 3 2 

37 1 0 5 

2 0 5 

3 0 5 0 0 

Positive Control 2-aminoanthracene 1.0 μg per plate 

1 635 1 

2 329 1 

 3 363 1 442 168 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 28 

Bacterial Mutation Assay 

Table 8 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA100 Experiment No : B1 

Activation Condition : None Cells Seeded : 0.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 190 1 

2 153 1 

3 195 1 179 23 

3 1 200 1 

2 205 1 

3 237 1 214 20 

6 1 192 1 

2 151 1 

3 194 1 179 24 

8 1 202 1 

2 167 1 

3 183 1 184 18 

11 1 225 1 

2 216 1 

3 207 1 216 9 

21 1 219 1 

2 185 1 

3 212 1 205 18 

37 1 0 5 

2 0 5 

3 0 5 0 0 

Positive Control sodium azide 1.0 μg per plate 

1 650 1 

2 630 1 

 3 567 1 616 43 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 29 

Bacterial Mutation Assay 

Table 9 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA100 Experiment No : B1 

Activation Condition : Rat Liver S9 Cells Seeded : 0.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 206 1 

2 227 1 

3 195 1 209 16 

3 1 220 1 

2 215 1 

3 213 1 216 4 

6 1 241 1 

2 176 1 

3 191 1 203 34 

8 1 235 1 

2 272 1 

3 201 1 236 36 

11 1 227 1 

2 242 1 

3 174 1 214 36 

21 1 291 1 

2 263 1 

3 247 1 267 22 

37 1 0 5 

2 0 5 

3 0 5 0 0 

Positive Control 2-aminoanthracene 1.0 μg per plate 

1 577 1 

2 550 1 

 3 1040 1 722 275 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 30 

Bacterial Mutation Assay 

Table 10 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1535 Experiment No : B1 

Activation Condition : None Cells Seeded : 1.0 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 17 1 

2 8 1 

3 6 1 10 6 

3 1 8 1 

2 10 1 

3 11 1 10 2 

6 1 11 1 

2 8 1 

3 9 1 9 2 

8 1 9 1 

2 13 1 

3 10 1 11 2 

11 1 7 1 

2 11 1 

3 7 1 8 2 

21 1 7 1 

2 5 1 

3 5 1 6 1 

37 1 0 5 

2 0 5 

3 0 5 0 0 

Positive Control sodium azide 1.0 μg per plate 

1 283 1 

2 310 1 

 3 286 1 293 15 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 31 

Bacterial Mutation Assay 

Table 11 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1535 Experiment No : B1 

Activation Condition : Rat Liver S9 Cells Seeded : 1.0 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 11 1 

2 7 1 

3 3 1 7 4 

3 1 14 1 

2 6 1 

3 2 1 7 6 

6 1 8 1 

2 8 1 

3 3 1 6 3 

8 1 6 1 

2 15 1 

3 8 1 10 5 

11 1 12 1 

2 6 1 

3 6 1 8 3 

21 1 6 1 

2 3 1 

3 5 1 5 2 

37 1 0 5 

2 0 5 

3 0 5 0 0 

Positive Control 2-aminoanthracene 1.0 μg per plate 

1 94 1 

2 39 1 

 3 86 1 73 30 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 32 

Bacterial Mutation Assay 

Table 12 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1537 Experiment No : B1 

Activation Condition : None Cells Seeded : 0.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 7 1 

2 8 1 

3 7 1 7 1 

3 1 2 1 

2 5 1 

3 3 1 3 2 

6 1 5 1 

2 1 1 

3 2 1 3 2 

8 1 5 1 

2 2 1 

3 8 1 5 3 

11 1 1 1 

2 0 1 

3 0 1 0 1 

21 1 1 1 

2 1 1 

3 1 1 1 0 

37 1 1 3 

2 0 3 

3 0 3 0 1 

Positive Control 9-aminoacridine 75 μg per plate 

1 919 1 

2 1161 1 

 3 896 1 992 147 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 33 

Bacterial Mutation Assay 

Table 13 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : TA1537 Experiment No : B1 

Activation Condition : Rat Liver S9 Cells Seeded : 0.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 7 1 

2 5 1 

3 9 1 7 2 

3 1 2 1 

2 5 1 

3 6 1 4 2 

6 1 4 1 

2 3 1 

3 1 1 3 2 

8 1 5 1 

2 3 1 

3 3 1 4 1 

11 1 9 1 

2 7 1 

3 4 1 7 3 

21 1 0 1 

2 0 1 

3 0 1 0 0 

37 1 0 3 

2 1 1 

3 0 3 0 1 

Positive Control 2-aminoanthracene 1.0 μg per plate 

1 40 1 

2 37 1 

 3 31 1 36 5 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 
BioReliance 

Study No. AC12HE.502700.BTL 34 

Bacterial Mutation Assay 

Table 14 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : WP2 uvrA Experiment No : B1 

Activation Condition : None Cells Seeded : 1.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 11 1 

2 17 1 

3 14 1 14 3 

3 1 14 1 

2 12 1 

3 12 1 13 1 

6 1 C 

2 13 1 

3 11 1 12 1 

8 1 10 1 

2 15 1 

3 9 1 11 3 

11 1 1 1 

2 5 1 

3 6 1 4 3 

21 1 0 1 

2 1 1 

3 5 1 2 3 

37 1 0 4 

2 0 4 

3 0 4 0 0 

Positive Control methyl methanesulfonate 1000 μg per plate 

1 146 1 

2 150 1 

 3 209 1 168 35 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 

C=Contamination 
BioReliance 

Study No. AC12HE.502700.BTL 35 

Bacterial Mutation Assay 

Table 15 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Strain : WP2 uvrA Experiment No : B1 

Activation Condition : Rat Liver S9 Cells Seeded : 1.7 x 108

Vehicle : Air Control Date Plated : 22-Apr-08 

Concentration 

mmoles/L Plate Number Revertants per plate 

Background 

Code Average Revertants 

Standard 

Deviation 

Vehicle 1 20 1 

2 12 1 

3 24 1 19 6 

3 1 31 1 

2 21 1 

3 17 1 23 7 

6 1 12 1 

2 19 1 

3 18 1 16 4 

8 1 6 1 

2 13 1 

3 20 1 13 7 

11 1 7 1 

2 NC 

3 5 1 6 1 

21 1 3 1 

2 5 1 

3 6 1 5 2 

37 1 0 4 

2 0 4 

3 0 4 0 0 

Positive Control 2-aminoanthracene 10 μg per plate 

1 202 1 

2 126 1 

 3 215 1 181 48 

Background Lawn Code 

 1=Normal; 2=Slightly reduced; 3=Moderately reduced; 4=Extremely reduced; 5=Absent 

 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate 

NC=No count due to procedural error in which the plate did not receive an aliquot of tester strain 
BioReliance 

Study No. AC12HE.502700.BTL 36 

Bacterial Mutation Assay 

 Summary of Results – Initial Mutagenicity Assay 

Table 16 

Test Article Id : 

Study Number : AC12HE.502700.BTL 

Experiment No : B1 

Average Revertants Per Plate ± Standard Deviation 

Activation Condition : None 

Dose (mmoles/L) TA98 TA100 TA1535 TA1537 WP2 uvrA 

Vehicle 35 ± 10 179 ± 23 10 ± 6 7 ± 1 14 ± 3 

3 27 ± 8 214 ± 20 10 ± 2 3 ± 2 13 ± 1 

6 28 ± 8 179 ± 24 9 ± 2 3 ± 2 12 ± 1 

8 22 ± 6 184 ± 18 11 ± 2 5 ± 3 11 ± 3 

11 19 ± 3 216 ± 9 8 ± 2 0 ± 1 4 ± 3 

21 4 ± 1 205 ± 18 6 ± 1 1 ± 0 2 ± 3 

37 0 ± 0 0 ± 0 0 ± 0 0 ± 1 0 ± 0 

Positive 129 ± 54 616 ± 43 293 ± 15 992 ± 147 168 ± 35 

Activation Condition : Rat Liver S9 

Dose (mmoles/L) TA98 TA100 TA1535 TA1537 WP2 uvrA 

Vehicle 24 ± 7 209 ± 16 7 ± 4 7 ± 2 19 ± 6 

3 15 ± 2 216 ± 4 7 ± 6 4 ± 2 23 ± 7 

6 25 ± 13 203 ± 34 6 ± 3 3 ± 2 16 ± 4 

8 18 ± 3 236 ± 36 10 ± 5 4 ± 1 13 ± 7 

11 13 ± 3 214 ± 36 8 ± 3 7 ± 3 6 ± 1 

21 3 ± 2 267 ± 22 5 ± 2 0 ± 0 5 ± 2 

37 0 ± 0 0 ± 0 0 ± 0 0 ± 1 0 ± 0 

Positive 442 ± 168 722 ± 275 73 ± 30 36 ± 5 181 ± 48 

Vehicle = Vehicle Control 

Positive = Positive Control (50 μL plating aliquot) 
BioReliance 

Study No. AC12HE.502700.BTL 37 

Table 17 

Confirmatory Mutagenicity Assay without S9 activation 

Study Number: AC12HE.502700.BTL Study Code: AC12HE 

Experiment: B2 Date Plated: 9/14/2010 

Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA100 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 40 13 0.4 54A 3, 37A 3, 28A 3 

11mmoles/L 88 77 0.9 45A, 42A, 176A 

8 mmoles/L 130 25 1.4 121A, 158A, 110A 

6 mmoles/L 113 18 1.2 107A, 98A, 133A 

3 mmoles/L 108 14 1.1 101A, 125A, 99A 

Untreated Air 96 12 110A, 86A, 92A 

TA1535 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 11 3 1.0 11A 3, 13A 3, 8A 3 

11mmoles/L 7 3 0.6 9A, 4A, 9A 

8 mmoles/L 11 4 1.0 8A, 15A, 11A 

6 mmoles/L 9 2 0.8 11A, 8A, 7A 

3 mmoles/L 8 1 0.7 8A, 7A, 9A 

Untreated Air 11 4 11A, 15A, 8A 

Key to Plate Postfix Codes 

4 

3 

Extremely reduced background 

Moderately reduced background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 
BioReliance 

Study No. AC12HE.502700.BTL 38 

Table 17 cont. 

Confirmatory Mutagenicity Assay without S9 activation 

Study Number: AC12HE.502700.BTL Study Code: AC12HE 

Experiment: B2 Date Plated: 9/14/2010 

Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA1537 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 1 2 0.2 3A, 0A, 0A 

11mmoles/L 2 1 0.4 3A, 3A, 1A 

8 mmoles/L 2 3 0.4 1A, 5A, 0A 

6 mmoles/L 8 2 1.6 9A, 9A, 5A 

3 mmoles/L 4 1 0.8 4A, 4A, 3A 

Untreated Air 5 3 8A, 3A, 5A 

WP2uvrA 37mmoles/L 8 4 0.2 7A, 4A, 12A 

21mmoles/L 15 3 0.3 13A, 19A, 13A 

11mmoles/L 33 6 0.7 40A, 28A, 31A 

8 mmoles/L 39 6 0.9 45A, 33A, 40A 

6 mmoles/L 55 7 1.2 48A, 61A, 57A 

3 mmoles/L 56 3 1.2 57A, 53A, 58A 

Untreated Air 45 9 37A, 54A, 45A 

TA100 SA 1.0 μg 772 20 8.0 749A, 784A, 784A 

TA1535 SA 1.0 μg 666 14 60.5 668A, 651A, 678A 

TA1537 9AAD 75 μg 515 62 103.0 576A, 517A, 452A 

WP2uvrA MMS 1000 μg 372 35 8.3 338A, 369A, 408A 

Key to Positive Controls Key to Plate Postfix Codes 

2NF 

SA 

9AAD 

MMS 

2-nitrofluorene 

sodium azide 

9-Aminoacridine 

methyl methanesulfonate 

4 

3 

Extremely reduced background 

Moderately reduced background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 
BioReliance 

Study No. AC12HE.502700.BTL 39 

Table 18 

Confirmatory Mutagenicity Assay with S9 activation 

Study Number: AC12HE.502700.BTL Study Code: AC12HE 

Experiment: B2 Date Plated: 9/14/2010 

Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA100 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 40 4 0.4 44A 2, 41A 2, 36A 2 

11mmoles/L 63 6 0.6 58A, 62A, 70A 

8 mmoles/L 72 10 0.7 73A, 82A, 62A 

6 mmoles/L 83 10 0.8 72A, 90A, 88A 

3 mmoles/L 88 5 0.8 86A, 94A, 84A 

Untreated Air 106 6 113A, 103A, 103A 

TA1535 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 7 2 0.8 8A 3, 8A 3, 4A 3 

11mmoles/L 7 4 0.8 4A, 11A, 7A 

8 mmoles/L 12 1 1.3 13A, 13A, 11A 

6 mmoles/L 5 4 0.6 5A, 8A, 1A 

3 mmoles/L 10 6 1.1 16A, 5A, 8A 

Untreated Air 9 3 7A, 12A, 8A 

TA1537 37mmoles/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21mmoles/L 5 4 0.7 9A, 5A, 1A 

11mmoles/L 5 2 0.7 3A, 7A, 4A 

8 mmoles/L 4 4 0.6 1A, 8A, 3A 

6 mmoles/L 6 2 0.9 8A, 7A, 4A 

3 mmoles/L 6 2 0.9 4A, 7A, 8A 

Untreated Air 7 2 8A, 5A, 8A 

TA100 2AA 2.0 μg 407 68 3.8 340A, 405A, 476A 

TA1535 2AA 1.0 μg 140 15 15.6 NCN#, 150A, 129A 

TA1537 2AA 1.0 μg 473 117 67.6 536A, 338A, 546A 

Key to Positive Controls Key to Plate Postfix Codes 

2AA 2-aminoanthracene 4 

2 

3 

NC 

N# 

Extremely reduced background 

Slightly reduced background 

Moderately reduced background 

No cells 

Not counted 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 
BioReliance 

Study No. AC12HE.502700.BTL 40 

Table 19 

Retest of Confirmatory Mutagenicity Assay without S9 activation 

Study Number: AC12HE.502700.BTL Study Code: AC12HE 

Experiment: B3 Date Plated: 9/28/2010 

Exposure Method: Plate incorporation assay Evaluation Period: 10/6/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 37 mmol/L 0 0 0.0 0M 5, 0M 5, 0M 5 

21 mmol/L 15 3 0.5 19M, 13M, 14M 

11 mmol/L 22 3 0.7 19M, 23M, 25M 

8 mmol/L 27 6 0.9 23M, 33M, 24M 

6 mmol/L 24 3 0.8 23M, 27M, 22M 

3 mmol/L 29 8 1.0 31M, 36M, 21M 

1 mmol/L 34 4 1.1 36M, 37M, 29M 

0.7 mmol/L 31 3 1.0 28M, 33M, 33M 

Untreated Air 30 10 25M, 41M, 24M 

TA1537 37 mmol/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21 mmol/L 4 1 0.4 4A, 5A, 4A 

11 mmol/L 6 5 0.6 3A, 4A, 12A 

8 mmol/L 7 5 0.7 12A, 3A, 7A 

6 mmol/L 11 3 1.1 13A, 8A, 11A 

3 mmol/L 10 2 1.0 8A, 9A, 12A 

1 mmol/L 6 4 0.6 1A, 7A, 9A 

0.7 mmol/L 6 3 0.6 3A, 8A, 7A 

Untreated Air 10 2 11A, CPN#, 8A 

TA98 2NF 1.0 μg 351 69 11.7 282A, 350A, 420A 

TA1537 9AAD 75 μg 633 142 63.3 721A, 469A, 710A 

Key to Positive Controls Key to Plate Postfix Codes 

2NF 

9AAD 

2-nitrofluorene 

9-Aminoacridine 

4 

CP 

N# 

5 

Extremely reduced background 

Contaminated plate 

Not counted 

Absent background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 
BioReliance 

Study No. AC12HE.502700.BTL 41 

Table 20 

Retest of Confirmatory Mutagenicity Assay with S9 activation 

Study Number: AC12HE.502700.BTL Study Code: AC12HE 

Experiment: B3 Date Plated: 9/28/2010 

Exposure Method: Plate incorporation assay Evaluation Period: 10/6/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 37 mmol/L 0 0 0.0 0M 4, 0M 4, 0M 4 

21 mmol/L 10 1 0.8 11M, 10M, 10M 

11 mmol/L 10 1 0.8 9M, 10M, 10M 

8 mmol/L 11 4 0.9 15M, 10M, 7M 

6 mmol/L 9 3 0.8 9M, 11M, 6M 

3 mmol/L 10 4 0.8 5M, 11M, 13M 

1 mmol/L 9 3 0.8 8M, 13M, 7M 

0.7 mmol/L 9 0 0.8 9M, 9M, 9M 

Untreated Air 12 1 13M, 11M, 11M 

WP2uvrA 37 mmol/L 4 1 0.1 3A 3, 5A 3, 5A 3 

21 mmol/L 34 13 0.8 49A, 28A, 24A 

11 mmol/L 32 4 0.8 32A, 29A, 36A 

8 mmol/L 35 6 0.8 41A, 29A, 34A 

6 mmol/L 28 5 0.7 27A, 24A, 34A 

3 mmol/L 43 6 1.0 37A, 44A, 48A 

1 mmol/L 42 5 1.0 48A, 38A, 41A 

0.7 mmol/L 39 15 0.9 53A, 42A, 23A 

Untreated Air 42 2 40A, 42A, 44A 

TA98 2AA 1.0 μg 408 78 34.0 318A, 459A, 448A 

WP2uvrA 2AA 10 μg 198 11 4.7 186A, 206A, 202A 

Key to Positive Controls Key to Plate Postfix Codes 

2AA 2-aminoanthracene 4 

3 

Extremely reduced background 

Moderately reduced background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 
BioReliance 

Study No. AC12HE.502700.BTL 42 

APPENDIX I: Historical Control Data 
BioReliance 

Study No. AC12HE.502700.BTL 43 

Historical Negative and Positive Control Values 

2005 – 2007 

revertants per plate 

Activation 

Strain Control None Rat Liver 

Mean SD Min Max Mean SD Min Max 

Neg 17 6 5 56 25 8 6 60

TA98 

Pos 189 112 30 1812 526 343 73 2669

Neg 133 30 52 250 143 32 67 263

TA100 

Pos 548 148 112 4349 698 343 232 2652

Neg 20 7 4 53 16 5 4 45

TA1535 

Pos 420 146 54 1019 104 59 18 985

Neg 7 3 1 20 8 3 1 29

TA1537 

Pos 740 356 14 2514 88 87 13 1297

Neg 21 9 5 69 23 10 4 65

WP2 uvrA 

Pos 214 137 28 1086 268 174 29 1178

SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control 

(including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); 

Pos=positive control 
BioReliance 

Study No. AC12HE.502700.BTL 44 

Historical Negative and Positive Control Values 

2007 – 2009 

revertants per plate 

Activation 

Strain Control None Rat Liver 

Mean SD Min Max Mean SD Min Max 

Neg 18 7 4 57 25 8 6 69 TA98 

Pos 221 131 34 1299 526 245 49 2342

Neg 132 32 51 255 141 35 56 268

TA100 

Pos 613 153 226 1837 776 380 224 3206

Neg 17 7 3 58 15 5 1 49

TA1535 

Pos 456 159 33 1601 120 85 18 1216

Neg 8 4 0 28 8 4 1 41

TA1537 

Pos 1040 576 24 4814 102 156 13 2360

Neg 28 11 6 72 31 12 5 77

WP2 uvrA 

Pos 344 163 44 1178 274 131 32 1656

SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control 

(including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); 

Pos=positive control 
BioReliance 

Study No. AC12HE.502700.BTL 45 

APPENDIX II: Study Protocol
BioReliance 

Study No. AC12HE.502700.BTL 46 
BioReliance 

Study No. AC12HE.502700.BTL 47 
BioReliance 

Study No. AC12HE.502700.BTL 48 
BioReliance 

Study No. AC12HE.502700.BTL 49 
BioReliance 

Study No. AC12HE.502700.BTL 50 
BioReliance 

Study No. AC12HE.502700.BTL 51 
BioReliance 

Study No. AC12HE.502700.BTL 52 
BioReliance 

Study No. AC12HE.502700.BTL 53 
BioReliance 

Study No. AC12HE.502700.BTL 54 
BioReliance 

Study No. AC12HE.502700.BTL 55 
BioReliance 

Study No. AC12HE.502700.BTL 56 
BioReliance 

Study No. AC12HE.502700.BTL 57 
BioReliance 

Study No. AC12HE.502700.BTL 58 
BioReliance 

Study No. AC12HE.502700.BTL 59 
BioReliance 

Study No. AC12HE.502700.BTL 60 
BioReliance 

Study No. AC12HE.502700.BTL 61 

APPENDIX III: Information for Japanese Regulatory Agencies
BioReliance 

Study No. AC12HE.502700.BTL 62 

Report of Results of Reverse-Mutation Assay in Bacteria 

1. Tester Strains 

(1) Procurement 

Strain Obtained from Date obtained 

 Date inspected 

the strain lot 

in storage 

TA98 

TA100 

TA1535 

TA1537 

TA1538 

TA97 

TA102 

Dr. Bruce Ames’ 

designated distributor, 

Discovery Partners 

International, San Diego, 

California 

13 November 

2002; 

16 January 2004 

(TA100 only) 

WP2 uvrA 

WP2 uvrA 

(pkM101) 

1 July 1987 

WP2 (pKM101) 

National Collection of 

Industrial and Marine 

Bacteria 

Aberdeen, Scotland 19 February 1993 

The genetic 

markers for each 

culture are 

confirmed on the 

day of use 

(2) Storage 

Freezing method Large quantity 

Storage temperature -60°C or colder 

Bacterial suspension 1.0 mL 

Composition DMSO 0.09 mL 
BioReliance 

Study No. AC12HE.502700.BTL 63 

2. S9 Mix 

(1) Source, Storage Temperature, etc. of S9 

Prepared on Used in Experiment No. 

29 January 2008 (Lot 2242) A1 and B1 Purchased from 

MolTox 

29 June 2010 (Lot 2639) B2 and B3 

Storage 

temperature -60°C or colder Name and model of storage apparatus 

So-Low, Model 

PR120-9 

(2) Preparation of S9 

Animal used Inducing substance 

Species, Strain Rattus norvegicus, Sprague 

Dawley Name Aroclor 1254 

Sex Male Administration 

method intraperitoneal 

Age (in weeks) Unknown (Lots 2242 and 2639)

Weight Unknown (Lots 2242 and 2639)

Administration 

period and 

amount (g/kgweight) 

single dose at 

0.5 gm/kg body 

weight, 5 days 

prior to sacrifice 

3. Preparation of Test Article Solution 

Solvent used 

Name Manufacturer Lot No. Grade and/or 

Purity (%) 

Not Applicable Not Applicable Not 

Applicable 

Not 

Applicable 

 Stability of test article in the 

solvent Unknown 

 Method of suspension when 

test article is difficult to 

dissolve 

Not applicable 

 Stability, storage time and 

temperature of formulation 

from preparation until use 

No change in appearance was noted in the formulations 

during the period of use (2 hours at room temperature) 
BioReliance 

Study No. AC12HE.502700.BTL 64 

4. Conditions of Pre-culture 

Nutrient broth Name Manufacturer Lot Nos. 

Oxoid Nutrient 

Broth No. 2 Oxoid Ltd. 544599, 847654 

and 876774 

 Period of pre-culture 12±2 hours 

 Storage time and temp. from 

inoculation to beginning of 

shaking culture 

<5 hours at ambient temperature 

 Storage time and temp. from 

end of culture to use for test <12 hours at 2-8°C 

 Model and manufacturer of 

shaker New Brunswick Scientific, model G-24 

 Method of shaking 

 (shaking type, speed, etc.) Rotary (125 rev/min.) 

 Culture vessel (shape, 

capacity) shape: cylinder, 200 mL 

 Culture volume 50 mL 

 Volume of inoculum 1 colony or 1 to 2 pellets 
BioReliance 

Study No. AC12HE.502700.BTL 65 

5. Agar Plate Medium 

(1) Top agar 

Name BBL Select 

Manufacturer Becton Dickinson 

Agar Lot No. 7078979 

7253113 

9265193 

(2) Minimum Glucose Agar 

Name BBL Select 

Manufacturer Becton Dickinson 

Lot No. 7253113 

Batch 

No. Preparation Date Used in Experiment No. 

16933 A1 

Made in-house Agar 

16934 

07 April 2008 

B1 

Name BBL Select 

Manufacturer Becton Dickinson 

Lot Nos. 7078979 

9265193 

0223574 

Batch 

No. Preparation Date Used in Experiment No. 

32323 18 March 2008 B1 

36101 12 August 2010 B2 

Agar 

36238 08 September 2010 B3 

Purchased from Moltox 

Volume of agar plate medium 25 mL 
BioReliance 

Study No. AC12HE.502700.BTL 66 

6. Test Results - Judgement of the results 

 Judgement Negative 

Reason for judgement and referential matters: 

No positive mutagenic response was observed with any of the tester strains in either the 

presence or absence of Aroclor-induced rat liver S9. 

Stability data were not provided for either lot/batch of the test article. In the absence of 

stability data, the Study Director has concluded that the test article was negative with the 

caveat that the maximum required concentration may not have been achieved due to 

instability or impurities. 

At the request of the Sponsor, dosing formulation analyses for concentration and stability 

were not conducted. Due to the lack of dose formulation analysis, the interpretation of the 

study data were based on the nominal dose levels as documented in the study records and 

not on the actual formulated test article concentrations as confirmed by analytical analysis. 

Nevertheless, toxicity in the assay demonstrated that the test system was dosed up to the 

regulatory required level. 

Referential matters 

The vehicle and positive control values indicate that all tester strains were functioning 

correctly and were capable of detecting a mutagen. 
BioReliance 

Study No. AC12HE.502700.BTL 67 

APPENDIX IV: Test Article Characterization
BioReliance 

Study No. AC12HE.502700.BTL 68 
BioReliance 

Study No. AC12HE.502700.BTL 69